The role of T cell phenotype in CAR-mediated cytotoxicity

Despite the clinical success of Chimaeric Antigen Receptor (CAR) therapy in haematological cancer, the mechanisms driving CAR-mediated T cell responses are not fully understood. There is very little evidence on the role of effector T cell phenotype in therapies relying on re-directed cytotoxicity, which commonly use bulk T cell populations. Since T cells can execute a plethora of functions in response to physiological TCR stimulation, we hypothesised that their phenotype directly affects the cytotoxic and immunomodulatory response upon CAR activation.

We created a model 2nd generation CAR construct and developed an in vitro assay allowing for the assessment of CAR-mediated cytotoxicity and activation in human T cell subsets. With these tools, we confirmed previous findings showing that CD4 CAR T cells are capable of target-mediated killing in a calcium-dependent manner and that the quality and quantity of released cytokines differs between CD4 and CD8 subsets.

Furthermore, we compared Th0, Th1 and Th2 CD4 CAR T populations regarding their cytotoxic and immunomodulatory potential and discovered that they all kill target cells in response to CAR stimulation. We also revealed significant differences between Th1 and Th2 cells in granzyme A and granzyme B levels as well as in FasL and TRAIL upregulation after TCR or CAR-mediated stimulation. Furthermore, we established that CAR induces the production and secretion of signature Th1 and Th2 cytokines, as well as other pro- and anti-inflammatory mediators. This extensive analysis provided new evidence on the differences in the cytotoxic and immunomodulatory potential of different Th CD4 CAR T populations.

Together with our collaborators from MedImmune (now Astra Zeneca), we developed a novel, genetically engineered mouse strain that allows for tracing of perforin expression in viable cells. We confirmed the co-expression of the mKATE2 reporter in relevant cell subsets and performed validation of protein expression. We then used this model to discover the dynamic changes of perforin expression in different subsets of T cells upon repeated TCR stimulation and to identify cells with cytotoxic potential. This is the first reliable tool for analysing and sorting murine cells which produce perforin at the protein level.